This proposal is concerned with the structure and function of the two distinct replication origins in mitochondrial DNA from malignant mouse and human tissue culture cells. The primary origin consists of a displacement loop triplex structure which contains a discrete family of initiation sequences hydrogen bonded at the origin of replication. We will use pulse-chase labeling and direct chemical sequencing methods to decipher the interrelationship between these sequences and their role in DNA replication. The secondary origin will be sequenced to help determine the control mechanism which results in sequential initiation at these two unique locations on the genome. We have recently established a transcriptional map of mouse mitochondrial DNA and it is now possible to study the details of how mitochondrial RNA transcripts are processed. The use of hybridization, restriction enzyme and gel electrophoresis techniques will allow us to determine the size and position of RNA processing intermediates. The number and location of promoter regions will be determined by the UV mapping approach. In particular, we will determine to what degree mammalian mitochondria utilize eukaryotic mechanisms of transcription such as splicing of transcripts. We hope to identify the structural gene products encoded by mitochondrial DNA by in vitro translation of isolated mitochondrial messenger RNA.